The hepatic peptide hormone hepcidin plays a central role in body iron metabolism. Despite its promise as a biomarker, the availability of high-sensitive hepcidin assays is still limited. We developed and validated a RadioImmunoAssay (RIA) to measure hepcidin quantitatively in human serum. This assay exhibited a very low detection limit (0.02 microg/l), low imprecision (coefficient of variation-range 4.4-6.2%) and good linearity and recovery (range: 81-105%). Hepcidin levels of samples of controls and patients with iron deficiency and inflammation showed an excellent correlation with our previously described quantitative time-of-flight mass spectrometry assay (range 2.5-266.8 microg/l, r = 0.92, P < 0.0001). The RIA detected: (i) differences in mean hepcidin levels between men (n = 29) and women (n = 35), (ii) differences between individuals of different HFE-genotypes (n = 60) and (iii) daily increases in hepcidin levels (n = 64). The assay (i) is easy to perform and many samples can be processed within one assay-run, (ii) shows accurate, reproducible and high-sensitive measurements and (iii) is anticipated to be particularly useful to study the effects of pathological and physiological stimuli on hepcidin levels in the lower range.